The overall goal of this proposal is to analyze the functional role of bone morphogenetic protein-2 (BMP2), a pleiotropic signaling molecule, and its downstream pathways in the control of uterine function during implantation. Our recent studies revealed that mice deficient in uterine BMP2 are infertile and exhibit a severe defect in stromal decidualization, a prerequisite for successful pregnancy. We also found that siRNAmediated downregulation of BMP2 expression in primary cultures of stromal cells isolated from pregnant mouse uterus efficiently blocked the differentiation process. To analyze the role of BMP2 during decidualization, we formulated the following specific aims: 1. Analyze the mechanisms by which the BMP2 signaling pathway controls stromal differentiation. Using microarray-based gene expression profiling, we identifed potential downstream targets of BMP2 in the uterus during decidualization. We will evaluate the functional contribution of selected BMP2 target genes in stromal cell differentiation by siRNA-mediated blockade of their expression and elucidate the signaling pathways of BMP2 in primary stromal cultures. 2. Investigate the function of Wnt 4 in murine uterine function. We identified Wnt 4 as a downstream target of BMP2 regulation in stromal cells undergoing decidualization. Attenuation of Wnt 4 expression by siRNAs greatly reduced stromal differentiation in vitro, suggesting that it is a candidate mediator of BMP2-induced decidualization. We will generate a conditional knockout of Wnt 4 to confirm our in vitro findings, and to analyze the functional consequences of this loss-of-function mutation during implantation. 3. Investigate the function of the BMP2 pathway in human decidualization. The expression of BMP2 is markedly induced in human endometrial stromal cells undergoing decidualization in vitro in response to steroid hormones and cAMP. Addition of exogenous BMP2 to these cultures also led to a robust enhancement of Wnt 4 expression and stimulated the differentiation process. We will evaluate the role of Wnt 4 in human stromal cell differentiation and identify additional gene pathways mediating BMP2 function in human stromal cells using microarray analysis. Of particular interest would be the factors regulated by BMP2 in both mouse and human endometrium. Collectively, the proposed experiments will test the hypothesis that BMP2 and its downstream signaling pathways play critical and conserved roles during decidualization and implantation.